When embedded in collagenous gel and implanted into animal tissue, endothelial cells can form networks and integrate with the host vasculature. Although this system has great potential for clinical applications, it is not yet sufficiently robust. A major reason for this is failure of the engrafted cells to connect with the host blood vessels through anastomosis, and very little is known about this process. Using longitudinal intravital imaging, we identified the mechanism responsible for the connections, and determined the rate-limiting step that slows the perfusion. The mechanism of vessel anastomosis involves wrapping of existing vessels by the engrafted cells, disruption of the host vessel wall and endothelial junction reassignment. The process allows the nascent vessels to tap into the pre-existing network. The proposed project will accelerate this process by specifically targeting components that hinder tapping, including basement membrane (Aim 1), host pericytes (Aim 2) and host endothelium (Aim 3). PUBLIC HEALTH RELEVANCE: Although it's an essential step for survival of transplanted or engineered tissue, very little is known about how blood vessel networks in the graft connect with host vessels to achieve perfusion. By observing the formation of these connections using intravital imaging, we found that the implanted vessels connect by wrapping around host vessels and degrading them to steal the blood flow. In the proposed work we will take advantage of our knowledge of this process to accelerate perfusion of grafts.